What effect do SDS, NP-40, Triton X-100, and lipids have on the sample preparation procedure?
The standard lysis buffer recommended for use with 2-D DIGE labelling contains 4% CHAPS. However, proteins for certain sample types may be more efficiently solubilized by the use of alternative detergents. Some of these detergents have been shown to have a detrimental effect on CyDye™ DIGE Fluor minimal dye labelling efficiency. Labelling efficiencies can be reduced by the addition of 1% SDS, but 0.25% appears to have no effect. NP40 is compatible to 1%. However Triton X-100 at a concentration of 1% has been shown to reduce labelling by 17%. Lipids may be a problem but these affect protein solubility rather than labelling efficiency. Always handle samples on a case-to-case basis. Test any non-standard lysis buffer components for their effect on labelling efficiency using 1-D gel analysis prior to 2-D DIGE experiments (see the Ettan DIGE System User Manual for detailed protocols).
